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2012-12-13

The Pre-Application of Hydrocortisone Cream and Its Effect on The Pre-Application of Hydrocortisone Cream and Its Effect on

Transdermal Drug Delivery by Phonophoresis Transdermal Drug Delivery by Phonophoresis

Patrick Thomas Webb Brigham Young University - Provo

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The Pre-Application of Hydrocortisone Cream and Its Effect on

Transdermal Drug Delivery by Phonophoresis

Patrick T. Webb

A thesis submitted to the faculty of Brigham Young University

in partial fulfillment of the requirements for the degree of

Master of Science

Gary Mack, Chair Aaron Wells

Dennis Eggett

Department of Exercise Sciences

Brigham Young University

December 2012

Copyright © 2012 Patrick Webb

All Rights Reserved

Abstract

The Pre-Application of Hydrocortisone Cream and Its Effect on Transdermal Drug Delivery by Phonophoresis

Patrick Webb Department of Exercise Sciences, BYU

Master of Science

Context: Transdermal delivery of hydrocortisone by phonophoresis is used for the treatment of musculoskeletal conditions. Research shows hydrocortisone and other white or opaque topical preparations transmit ultrasound energy poorly. Effective transmission of ultrasound is important in phonophoresis. Main Outcome measured: Samples of subcutaneous interstitial fluid were collected during and for 20 minutes following phonophoresis treatment. Cortisol concentrations were analyzed by an enzyme linked immune-assay (ELISA) test. Objective: Determine the subcutaneous cortisol concentration after two different phonophoresis treatments using a 2.5% hydrocortisone preparation. Design: Randomized design in which 22 healthy participants were assigned to receive a phonophoresis treatment where: 1) hydrocortisone cream was rubbed in completely prior to phonophoresis or 2) hydrocortisone powder was compounded with an ultrasound coupling gel. Test Subjects: 22 healthy individuals were recruited: 13 females with a mean age of 21 years and 9 males with a mean age of 21.8 years. Intervention: Phonophoresis consisted of pulsed ultrasound at 1 MHz, 1.0 w/cm2, and a 50% duty cycle. The treatment duration was 10 minutes and was localized over the distal gastrocnemius muscle. Results: We observed no significant difference in subcutaneous cortisol concentration between the two phonophoresis treatments (p=0.05). Also no significant difference was detected between pre and post-treatment cortisol levels within each individual treatment group. Conclusions: Our data indicates that completely rubbing a topical hydrocortisone application into the skin prior to placement of ultrasound gel does not result in increased transdermal delivery of cortisol when compared with the use of a compound of ultrasound gel and hydrocortisone powder applied topically to the skin. Key words: phonophoresis, hydrocortisone, microdialysis

Acknowledgements

Thanks to those who have served as my graduate advisers and committee members.

Especially David Draper, Aaron Wells and Dennis Eggett for all the time they have spent in

helping me carry out this research project. I would like to thank Dr. Wells for helping me to

develop my ideas into a valuable research project and for encouraging me to make sure it is

“Done Right.”

Thanks to my mom for her constant support and for being willing to help me out in any

way possible. I want to thank my dad for encouraging me to do my best work throughout my life

and making sure that I knew that “A job worth doing is worth doing well.”

Lastly, thanks to the BYU students and faculty, family members and friends as well as so

many others who have helped me. There have been many.

iv

Table of Contents

List of Tables .......................................................................................................................v

List of Figures .................................................................................................................... vi

Introduction ..........................................................................................................................1

Methods................................................................................................................................3

Results ..................................................................................................................................8

Discussion ............................................................................................................................8

Clinical Implications ..........................................................................................................12

References ..........................................................................................................................14

v

List of Tables Table:

1. Study Measurement Values………………………………..…………………..17

vi

List of Figures Figure:

1. Guide Cannula needle ...................................................................................................... 18

2. Doppler ultrasound Measuring Probe Depth ................................................................... 19

3. Phonophoresis Treatment................................................................................................. 20

1

Introduction

When localized delivery of medication is desired for the treatment of musculoskeletal

trauma, injection using a hypodermic needle is the most commonly used method.1 The use of

hypodermic needles, however, can result in pain or possible infection, thus decreasing patient

compliance to the procedure. Transdermal drug delivery is the administration of therapeutic

agents through intact skin.2 Transdermal drug delivery presents an alternative means of local

drug delivery. Phonophoresis is a form of transdermal drug delivery, where therapeutic

ultrasound is used to increase the transcutaneous transmission of drugs. Phonophoresis can be

used to introduce medication locally to a specific area without painful injections. Therapeutic

ultrasound is thought to augment transdermal drug delivery by modifying the permeability

characteristics of the skin.3 Increased skin permeability resulting from ultrasound has been

attributed to a phenomenon called cavitation. Cavitation refers to gas bubbles within the tissue

which are made to oscillate as a result of the penetrating ultrasound energy.3, 4 The oscillation of

these bubbles near the skin provokes disorganization through the lipid layers of the stratum

corneum. This disorganization is thought to create aqueous channels through the stratum

corneum and increase the permeability of the skin.4, 5

During phonophoresis, one of two pre-treatment protocols is commonly used.6 In the first

protocol a topical medication alone is applied to the skin. This topical medication serves as the

ultrasound coupling agent. The second pre-treatment protocol used in phonophoresis involves

mixing the topical medication into a substance that is intended for use as an ultrasound coupling

agent. Some topically applied pharmaceuticals; however, which are commonly used for

phonophoresis have shown poor conductivity such as those that might be opaque or thick in

consistancy.6 Cameron et al. also showed that adding a medium that transmits poorly in equal

2

parts to a medium that transmits ultrasound energy well did not improve transmission.6

A coupling medium should allow ultrasonic energy to enter the target tissue at the desired

intensity. Any attenuation of the ultrasound energy may decrease the effectiveness of

phonophoresis. Because of this, both of the above mentioned methods may compromise the

effectiveness of some phonophoresis treatments.

Phonophoresis is both non-invasive and allows for the local introduction of medication.1

An objective method of data collection was selected that would also fit those two criteria.

Microdialysis probes were utilized to collect samples of interstitial fluid local to the treatment

site which was used in quantifying drug delivery. The efficacy of pharmaceuticals is often based

on an association between observed affects and drug concentrations in the blood plasma.7, 8

Concentrations of drug molecules can differ significantly between blood and the tissues.9

Researchers have shown that only drug molecules in the target tissue are responsible for the

efficacy of a drug.10, 11 Unlike samples gathered through a blood draw; samples collected through

microdialysis probes come from interstitial fluid within the target tissue as opposed to circulating

blood plasma. Microdialysis is currently the most appropriate sampling technique that provides

for the analysis of drug molecules within a tissue.8, 12

The purpose of this study was to evaluate the effectiveness of phonophoresis treatments

in which hydrocortisone cream is rubbed completely into the skin prior to the phonophoresis

treatment using an ultrasound gel couplant. No researchers have specifically addressed the

efficacy of thoroughly applying a thick, white topical medication similar to hydrocortisone

preparations into the skin before the ultrasound gel is applied on top of the skin.

3

Methods

Study Design

The 2x2 design of this study includes 2 treatment groups and 2 covariates. The treatment

groups are as follows: 1) a 2.5% hydrocortisone cream was rubbed into the skin for 5 minutes

followed by application of an ultrasound gel and phonophoresis treatment, and 2) a 2.5%

hydrocortisone powder compounded in ultrasound gel was applied to the skin in preparation for a

phonophoresis treatment.

The depth of the microdialysis probe as well as the test subject’s pre-treatment cortisol

levels were measured and used as covariates. Volunteers with recent or current injury to the

lower extremity, history of general illness or decreased sensation to lower extremities were

excluded from participation. Success of the phonophoresis treatment or transdermal drug

delivery was assessed objectively by measuring the difference in cortisol concentrations in the

dialysate collected from interstitial fluid both prior to and following the treatment.

Subjects

There were 22 healthy individuals recruited as subjects and tested during this study: 13

females (mean age of 21 years) and 9 males (mean age of 21.8 years.) All subjects were college

students from Brigham Young University. Subjects received treatment from one of two treatment

groups to which they were randomly assigned. Prior to participation in this study all volunteers

were required to read and sign the Institutional Review Board approved consent form.

4

Instruments

Ultrasound Unit: The ultrasound treatment was applied with an Omnisound 3000

Ultrasound device (Accelerated Care Plus, Reno, NV) with a 7.2 cm diameter sound head and a

5 cm2 crystal at an intensity of 1.0 W/cm2 with a 50% duty cycle.

Aquasonic 100 water-soluble hypoallergenic ultrasound transmission gel (Parker

Laboratories, Inc, Fairfield, NJ) was used as the ultrasound coupling agent in the pre-application

of topical medication treatment group.

A hydrocortisone cream with a 2.5% concentration (Fougera A division of Nycomed US

Inc. Melville, New York 11747) was used.

Hydrocortisone powder with a 2.5% concentration from Medisca Chemical, Plattsburgh

NY for use in this study. This compounding of the U.S gel and Hydrocortisone powder was

prepared by a pharmacist at the Brigham Young University Student Health Center Pharmacy and

used in group #2.

A Model LogiQ 5e, Doppler imaging ultrasound (General Electric Company, Fairfield,

CT) was used to measure the depth of the microdialysis probe subcutaneously following

insertion.

A cortisol high sensitivity ELISA kit (catalog #CO194S) from CalBioTech (A Life

Sciences Company, 10461 Austin Drive suite G, Spring Valley, CA USA.) was used for the

chemical analysis of the collected samples.

Tissue Perfusion

Sterile saline was perfused through the microdialysis probes using a Harvard Apparatus

PHD 2000 Programmable Infusion Pump (Harvard Apparatus, Holliston, MA).

5

Probe construction

The microdialysis probes used in this study were constructed in a similar manner to those

used in previous research involving transdermal drug delivery.13 One difference was employed

during the construction of the microdialysis probes for this study. In order to approximate

clinical protocols for ultrasound use in our study a template was made to ensure that the

ultrasound treatment performed on each test subject covered an area no larger than twice the

diameter of the sound head on our therapeutic ultrasound unit. To more closely match the length

of the treatment area in this study the hollow-fiber section in the microdialysis probes was

increased from 2.5 cm to 5 cm.

Probe placement

The treatment site in this study was located on a skin site over the musculotendonous

junction of the Gastrocnemius muscle and the Achilles tendon on the left leg of each research

subject. (See Figure 1) This area was cleansed and sterilized with a Provadone swab. The

microdialysis probe was then inserted subcutaneously. A 27-gauge needle was used as a guide

cannula for microdialysis probe placement. The guide cannula was inserted under the skin in-line

with the long axis of the gastrocnemius muscle. Early clinical trials in humans showed

microdialysis probes successful in collecting samples in the subcutaneous adipose tissue.14

The cannula entrance and exit sites in the skin were separated by approximately 6.0 cm.

This length was meant to approximate the size of the template which was used to create a

uniform treatment area for each ultrasound treatment. Following insertion of the needle or guide

cannula its depth and the depth at which the probe would rest throughout the study protocol was

measured and recorded with a Doppler imaging ultrasound.

6

Mean probe depth was 0.3118 cm ± 0.09. The microdialysis probe was threaded through the

inside of the guide cannula which was when removed.

To ensure that the cortisol detected in the interstitial fluid passed transdermally and to

avoid contamination of the samples of dialysate collected, the entrance and exit portals for the

microdialysis probes were sealed to the skin using a Tegaderm patch.

Drug Delivery

After placement, the microdialysis probes were perfused with 0.9% sterile saline at a rate

of 10µl/min for 60 minutes using an infusion pump.13 The purpose of this period was to allow

the tissues to recover from the trauma of the needle insertion and for any elevated physiologic or

inflammatory responses to return to base-line. At minute 60 of the recovery period the infusion

rate was adjusted to 2 µl/min and a sample was collected for 30 minutes to determine pre-

treatment tissue cortisol levels. The perfusion rate was maintained at 2 µl/min for the remainder

of the study protocol.

Prior to the ultrasound treatment the test subjects in each of the two treatment groups

received a different preparation. Two milliliters of the 2.5% hydrocortisone cream was rubbed

into the skin of the 11 test subjects in group one for 5 minutes. Following those 5 minutes the

excess cream was removed and 3 ml of water-based ultrasound gel was then applied to the skin.

Test group two had 5 ml of a prepared compound of 2.5% hydrocortisone powder and ultrasound

gel applied to the treatment site. Both of these were followed by a 10 minute ultrasound

treatment to complete the phonophoresis. During the ultrasound treatment dialysate from the

subcutaneous microdialysis probe was collected from the treatment site for analysis. The

parameters for the ultrasound treatment were the same for all study subjects: 1 MHz, 1.0 w/cm2,

50% pulsed duty cycle. A pre-fabricated template was placed on the skin of each research subject

7

to ensure consistency in the ultrasound treatment and confine the treatment area to 2 times the

size of the ultrasound head.

At the conclusion of the 10 minute phonophoresis treatment, any remaining water-based

gel (in test group 1) or combination of water based gel and hydrocortisone powder (in test group

2) was carefully removed from the treatment site. Subjects remained in a prone position for an

additional 20 minutes while dialysate collection continued. Thirty minutes after the ultrasound

treatment was initiated the saline perfusion was discontinued and the dialysate collected.

Following the 120 minutes of the designed study protocol the ultrasound template and the

subcutaneous microdialysis probe was removed. The portal sites was again cleansed with alcohol

and covered. The collection vials were removed, labeled, and stored in a freezer at -20° C for

later analysis.

Statistical Analysis

The data collected from each group was analyzed using an ANCOVA to determine

whether one treatment method yielded a greater change in post-treatment cortisol levels.

Analysis was also done to compare the pre-treatment and post-treatment cortisol concentrations

in each individual group to determine the effectiveness of the phonophoresis treatment.

The measured depth of the microdialysis probes and pre-treatment dialysate cortisol

concentrations were used as covariates. Cortisol levels fluctuate between individuals and even

throughout the day in the same individual in response to many variables. Because the difference

in cortisol levels within the body of each test subject the pre-treatment cortisol levels for each

individual were recorded and used as another covariate in the data analysis. For our statistical

analysis the level of significance was set at P ≤ 0.05.

8

Results

In treatment group #1 the change in dialysate cortisol concentration after the

phonophoresis was 13.2± 67.7 ng/ml; not significantly different from zero (t19=.96, p=.35). In

treatment group #2 the change in dialysate cortisol concentration after the phonophoresis was

-15.7 ± 30.3 ng/ml, also was not significantly different from zero (t19= -1.14, p=.27). Overall

there were no significant differences in post-treatment dialysate cortisol levels between treatment

groups. Nor was there a significant difference between pre and post-treatment dialysate cortisol

concentrations in either of the individual treatment groups.

The pre-treatment, post-treatment, and change in dialysate cortisol concentrations were

similar in both treatment groups (See Table 1). However, the pre-treatment dialysate cortisol

levels significantly influenced the magnitude of the change in dialysate cortisol levels (P=0212.)

Specifically, when higher values for pre-treatment dialysis cortisol concentration were seen, a

smaller increase in post-treatment dialysis cortisol concentration could be expected.

Microdialysis probe depth was not found to contribute significantly to the magnitude of the

change in dialysate cortisol levels (p = 0.9735), and was subsequently removed from the analysis

model.

Discussion

Research has found the hydrocortisone to be a poor conductor of ultrasound energy both

in the common OTC preparation which is thick and white and when mixed in solution with

ultrasound couplant gel.6 This led, in part, to our hypothesis and study design to first topically

apply the hydrocortisone prior to the application of the ultrasound gel so that only the coupling

gel was present between the soundhead and the skin during the ultrasound treatment.

9

The success of phonophoresis has been reported by researchers for the treatment of

musculoskeletal conditions, although some of these studies based their conclusion on subjective

data. 13, 14-17 This subjective data may be collected through an increase in function or a decrease

in pain communicated by a visual analogue scale. The term Clinical Evaluation also has been

used to describe methods by which subjective data have been gathered in studies which reflect

positively on phonophoresis. In 2005 Pribicivic and Pollard reported the success of a multi-

modal treatment for shoulder impingement which involved a phonophoresis treatment with 1

percent hydrocortisone cream. 18 In other research involving phonophoresis, chemical analysis of

either blood samples or tissue biopsies was used as a means of collecting more objective data.

Older studies where chemical analysis was used to gather data reported successful phonophoresis

treatments. 19, 20 More recent studies where a chemical analysis was employed seem to trend

toward a negative outcome in the evaluation of phonophoresis. 21, 22, 23

In our study a chemical analysis was used to evaluate the cortisol concentrations found in

samples of interstitial fluid. By this method, the success of the phonophoresis or transdermal

drug delivery of cortisol could be objectively evaluated. The results of our study, when

compared to other research.21, 22, 23 support the hypothesis that phonophoresis and hydrocortisone

cream is a poor method for delivery of cortisol to underlying tissue.

No significant difference was found in the change in dialysate cortisol concentration

between the two treatment groups. These results do not support the idea that rubbing a

hydrocortisone cream into the skin prior to the application of ultrasound gel is more effective for

phonophoresis than simply mixing hydrocortisone cream with ultrasound gel. Also, at the

beginning of this study the assumption was made that the phonophoresis treatments would be

effective in the transdermal delivery of cortisol. Because no significant change was seen

10

between the pre and post treatment dialysate cortisol levels in either of the two treatment groups

the results of this study do not support this assumption that phonophoresis effective in the

transdermal delivery of the hydrocortisone.

Hydrocortisone was the topical medication used in this study because of its thick, white

or opaque preparation. Cortisol is a naturally occurring substance in the body and fluctuations of

this chemical inside the human body as a result of a multidude of factors may have limited our

ability to collect a sample and accurately measure a baseline concentration of tissue cortisol.

“Although microdialysis is a minimal invasive technique it is obvious that insertion of the

probe will increase the local blood flow.” 24 In 1998 Peterson reported an increase in histamine

levels induced by the trauma caused by insertion of a microdialysis probe.25 In our study, as has

previously been done26, 27 a recovery period (60 minutes) after the insertion of the probe designed

to allow the local tissue to return to a homeostasis after the trauma of the probe insertion. One

potential limitation of our study may be that the recovery time after the insertion of the cannula

needle was insufficient to allow the underlying tissue to return to a normal physiologic state.

Some of the base-line cortisol measurements in our study were greater than those cortisol

measurements taken post-treatment. The elevated pre-treatment tissue cortisol levels may

indicate the presence of local “unresolved” trauma.

In a study by Lee, microdialysis probes were used for data collection in the dermis. The

microdialysis probes were inserted at a depth of approximately 3 mm. The recovery period after

the insertion of the probe and before samples were taken was 150 minutes in length. Skin blood

flow directly over the microdialysis probe was measured to ensure that the initial inflammatory

or physiologic response to the placement of the probe had returned to baseline.28 An increase of

both local blood flow 24 and histamine25 has been addressed in previous research; as a result of

11

the insertion of microdialysis probes in human skin. If a correlation between the two could be

shown then a more appropriate recovery time could possibly be verified during a study protocol

by using superficial blood flow as an index of recovery from the insertion trauma.

In 2011 Yeager explored the serum cortisol concentrations required to produce a systemic

anti-inflammatory effect in post-surgical (cardiopulmonary bypass) patients as characterized by

the concentration of plasma IL-6.29 Yeager describes ‘normal’ serum cortisol concentrations in

the range 15–20 ug/dl. It was found that an increase to a level of 35–45 ug/dl, a nominal 228%

increase from ‘normal’ serum cortisol levels, produced an anti-inflammatory effect. Pre-

treatment (normal) tissue cortisol concentrations measured in this study were 4.57 ug/dl and 4.16

ug/dl in Group 1 and Group 2, respectively, which are roughly one-third to one-fourth the normal

measured blood serum concentrations reported by Yeager.29 Caution needs to be exercised when

drawing parallels between serum cortisol levels and tissue concentrations. If we can extrapolate

from serum cortisol levels of the Yeager investigation to tissue cortisol levels measured in our

study, we would expect that an approximate two-fold increase tissue cortisol level would yield

any anti-inflammatory response. Our study produced a 29% increase in tissue cortisol levels for

Group 1, and an unexpected 35% decrease in cortisol levels for Group 2. Thus, extrapolating

from the Yeager study, neither of the two pre-treatment protocols for topical cortisone

application by phonophoresis investigated here appears to produce increases in tissue cortisol

concentrations that would result in an anti-inflammatory response. In fact, the average increase

in cortisol concentration in our study (29% in Group 1) is only roughly one-eighth the increase

required in serum cortisol concentration (228%) suggested by Yeager to produce an anti-

inflammatory effect.29

12

Future research using microdialysis probes to collect interstitial fluid to find a base-line

or pre-treatment measurement of cortisol should consider an increased recovery time after

insertion of the cannula needle and microdialysis probe. The insult to the skin resulting from the

insertion of the catheter needle may result in increased blood flow as well as other chemical

mediators produced and released by the body. An increase in recovery time after insertion of a

cannula needle may not be as important when using chemicals that are not naturally occurring in

the body. Verification of blood flow associated with any physiologic inflammatory response due

to the insertion of a microdialysis probe into the skin should be investigated in future studies.

Clinical Implications

The purpose of this study was to evaluate the effectiveness of a phonophoresis treatment

with hydrocortisone cream by first rubbing the cream completely into the skin prior to the

application of ultrasound through a gel couplant. Our data do not support the hypothesis that our

modified phonophoresis protocol, rubbing the hydrocortisone cream into the skin prior to the

application of ultrasound gel, would increase the effectiveness of the phonophoresis. Further the

data collected did not support the assumption that the phonophoresis protocol used in this study

would be effective in transdermal delivery of cortisol.

This study was designed with clinical protocols in mind. One study found that substances

used in phonophoresis that had a thick white preparation, including hydrocortisone, conducted

ultrasound energy poorly.6 It was also found that combining these same substances in equal parts

with a substance that has been shown to conduct ultrasound energy well did not result in an

increase in conductivity when compared with water.6 It was based off of this that a change in

pre-treatment protocol was suggested where the topical hydrocortisone would be rubbed into the

skin with any excess being removed prior to the application of ultrasound couplant gel so that

13

only a substance shown to conduct ultrasound energy well would be present between the sound-

head and the skin during the ultrasound portion of the phonophoresis. Although the theory

behind the suggested phonophoresis protocol technique adjustment seems sound no change in

clinical protocols can be suggested based off of this study. In addition the use of hydrocortisone

in phonophoresis should be reconsidered as a therapeutic modality for the treatment of

musculoskeletal conditions.

14

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15

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18. Pribicevic M, Pollard H. A multi-modal treatment approach for the shoulder: A 4 patient

case series. Chiro Osteopath. 2005;13:20.

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16

21. Kuntz AR, Griffiths CM, Rankin JM, Armstrong CW, McLoughlin TJ. Cortisol

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17

Table 1.

Study Measurement Values

Treatment

Group

Probe Depth

cm

Pre-Tx Dialysate

Cortisol

ng/ml

Post-Tx Dialysate

Cortisol

ng/ml

Change in Dialysate

Cortisol

ng/ml

#1 (HC) 0.320 ± 0.095 45.7 ± 48.7 57.9 ± 59.9 13.2 ± 67.7

#2 (Comp) 0.300 ± 0.093 41.6 ± 54.2 27.0 ± 39.4 -15.7 ± 30.3

Difference 0.02±0.002 -0.5 ± 0.2* 28.8 ± 19.5

Values represent the mean ± 1 SD for 11 subjects in each group.

18

Figure 1: Guide Cannula Needle

19

Figure 2: Doppler Ultrasound Measuring Probe Depth

20

Figure 3: Phonophoresis Treatment

.